Importantly, the assay can be performed at bedside and in rural areas
using only Selleck AZD0530 a water bath (Tomita et al., 2008). Several LAMP assays have been developed to detect common causative pathogens of BM such as Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Escherichia coli and Staphylococcus aureus (Seki et al., 2005; Yamazaki et al., 2008; Hanaki et al., 2011; Kim et al. 2011; McKenna et al. 2011). However, no LAMP assay has been reported to detect Streptococcus agalactiae and Streptococcus suis, which are two of the most common pathogens of BM in some countries (Mai et al., 2008; Chiba et al., 2009). Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a LAMP assay capable of detecting multiple bacterial species based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The broad range LAMP primers were designed to be specific for eubacterial 16S rRNA-specific gene. This gene was chosen because
of its highly conserved regions among species and has been widely used as a target for broad range PCR method (Gray et al., 1984; Lane et al., 1985). The partial nucleotide sequences of the 16S rRNA genes of S. aureus (GenBank FJ907240.1), S. pneumoniae (Z22807), S. suis (Z22776.1), S. agalactiae (Z22808), N. meningitidis (Z22806), H. influenzae (Z22809.1) and E. coli (AY513502.1) were PD0332991 chemical structure retrieved from the GenBank database and were aligned to identify potential target regions using multialin software (Corpet, 1988). Several conserved regions were chosen for designing of LAMP primer set using the LAMP primer design software Primer Explorer
version 4 (Eiken Chemical Co., Ltd, Tokyo, Japan). A set of four primers including two outer primers (forward primer F3 and backward primer B3) and two inner primers [forward inner primer (FIP) and backward inner primer (BIP)] that identified six distinct regions on the potential target sequence was designed. This study Aldol condensation was approved by the institutional ethical review committees of the Institute of Tropical Medicine, Nagasaki University. Serotypes 3 and 10 of S. pneumoniae were isolated from upper respiratory tract in Vietnamese patients. Two strains (8-01 and 8-02) of S. suis serotype 2, E. coli, S. aureus and S. agalactiae were also isolated from Vietnamese patients. In addition, H. influenzae and N. meningitidis were isolated from Japanese patients. The S. pneumoniae was cultured on rabbit blood Muller Hinton agar, while other bacteria were grown on rabbit blood brain heart infusion agar. Grown bacteria were harvested and suspended in normal saline. The cells were pelleted, suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.